Evaluation of epifluorescence image analysis of biofilm growth on stainless steel surfaces

Biofilm growth of Bacillus subtilis, Pseudomonas fragi, Pediococcus inopinatus and Listeria monocytogenes was studied on stainless steel surfaces at room and low temperatures to evaluate the results of traditional hygiene measures. The results were compared with those of image analysis of stainless steel surfaces in an epifluorescence microscope. Statistical analyses were carried out to determine the variations between the conventional cultivation swab method, the glycocalyx amount obtained using swabbing, and the values of the areas of the biofilm, slime and cells. As a general rule, old biofilms showed total counts at approximately the same levels as the young biofilm. The results showed that temperature affected the results for all strains except B. subtilis. The strains of Pe. inopinatus and Ps. fragi showed increased attachment at 6°C and L. monocytogenes at 25°C. The biofilm slime was more easily detached than the cells. The results indicated that the traditional swab method is not reliable for the measurement of biofilm formation on surfaces.     

Anaerobic reduction of a sulfonated azo dye, Congo Red, by sulfate-reducing bacteria

The capacity for anaerobic decolorization of a sulfonated azo dye, Congo Red, by a strain of a sulfate-reducing bacterium was evaluated. After optimizing the growth rate of the bacteria on a simple carbon source and terminal electron acceptor pair, lactate and sulfate, respectively, the effect of the dye concentration on their growth rate was analyzed. The decolorization rate was affected by the dye concentration in the growth medium. The azo-bond cleavage mechanism of reductive decolorization with the formation of benzidine was consistent with the results, as this metabolite was identified by high-performance liquid chromatography. Several fractions of the culture medium, including lysed cell extracts, were examined for the capacity to reduce the azo dye. This reduction capacity was found in the culture medium in which the cells had previously grown. The results showed that the mechanism of reductive decolorization of this sulfonated azo dye was extracellular and nonenzymatic, consistent with the production of sulfide anion by the microorganisms while growing on lactate and sulfate. The sulfide anions were the cause of the reduction leading to the disappearance of color in the medium. To increase the rate of decolorization, the presence of ferrous ion was also necessary together with the lactate and sulfate substrates.     

Spectroscopic evidence for triplet excitation energy transfer among carotenoids in the LH2 complex from photosynthetic bacterium <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis>

The LH2 complex from Rhodopsudomonas (Rps.) palustris is unique in the heterogeneous carotenoid compositions. The dynamics of triplet excited state Carotenoids (³Car* has been investigated by means of sub-microsecond time-resolved absorption spectroscopy both at physiological temperature (295 K) and at cryogenic temperature (77K). Broad and asymmetric T _n ←T ₁ transient absorption was observed at room temperature following the photo-excitation of Car at 532 nm, which suggests the contribution from various carotenoid compositions having different numbers of conjugated C=C double bonds (N_c=c). The triplet absorption bands of different carotenoids, which superimposed at room temperature, could be clearly distinguished upon decreasing the temperature down to 77 K. At room temperature the shorter-wavelength side of the main T_n04T₁ absorption band decayed rapidly to reach a spectral equilibration with a characteristic time constant of ∽1 μs, the same spectral dynamics, however, was not observed at 77 K. The aforementioned spectral dynamics can be explained in terms of the triplet-excitation transfer among heterogeneous carotenoid compositions. Global spectral analysis was applied to the time-resolved spectra at room temperature, which revealed two spectral components peaked at 545 and 565 nm, and assignable to the T_n04 T₁ absorption of Cars with Nc=c=11 and Nc=c=13, respectively. Surprisingly, the decay time constant of a shorter-conjugated Car, i.e. 0.72 ώs (aerobic) and 1.36 ώs (anaerobic), is smaller than that of a longer-conjugated Car, i.e. 2.12 us (aerobic) and 3.75 ώs (anaerobic), which is contradictory to the general rule of carotenoids and relative polyenes. The results are explained in terms of triplet-excitation transfer among different types of Cars. It is postulated that two Cars with different conjugation lengths coexist in an α, β-subunit in the LH2 complex.     

A switch-on fluorescence assay for bacterial β-lactamases with amyloid fibrils as fluorescence enhancer and visual tool

Herein is described the development of a novel switch-on fluorescence assay for detecting β-lactamases. The fluorescence assay comprises two components: solid beads coated with a β-lactam antibiotic, which is linked to an environment-sensitive fluorophore (dansylaminothiophenol, DTA), and amyloid fibrils of hen lysozyme (acting as fluorescence enhancer and visual tool). In the presence of the clinically significant TEM-1 β-lactamase, the DTA-antibiotic complex on the solid beads is hydrolyzed, thus releasing the DTA dye into solution. The DTA dye is only weakly fluorescent in solution but gives strong green fluorescence upon binding to lysozyme fibrils. These strongly fluorescent DTA-bound fibrils can be easily visualized by the naked eye upon illumination of the sample with a simple UV lamp. The fluorescence assay can detect TEM-1 at low concentration (0.01 nM). In contrast, no observable fluorescence appears when the fluorescence assay is performed on samples without the TEM-1 β-lactamase.     

Monitoring microbial populations of sulfate-reducing bacteria using an impedimetric immunosensor based on agglutination assay

An impedimetric immunosensor was fabricated for rapid and non-labeled detection of sulfate-reducing bacteria, Desulforibrio caledoiensis (SRB) by immobilizing lectin-Concanavalin A using an agglutination assay. The immobilization of lectin was conducted using amine coupling on the surface of a gold (Au) electrode assembled with 11-Mercaptoundecanoic acid. Electrochemical impedance spectroscopy (EIS) was used to verify the stepwise assembly of the sensor system. The work conditions of the impedimetric immunosensor, such as pH of the buffer solutions and the incubation time of lectin, were optimized. Faradic impedance spectra for charge transfer for the redox probe Fe(CN)₆^3−/4−were measured to determine SRB concentrations. The diameter of the Nyquist diagram that is equal to the charge-transfer resistance (R_ct) increased with increasing SRB concentration. A linear relationship between R_ct and SRB concentration was obtained in SRB concentration range of 1.8 to 1.8 × 10⁷ cfu/ml. The variation of the SRB population during the growth process was also monitored using the impedimetric immunosensor. This approach has great potential for simple, low-cost, and time-saving monitoring of microbial populations.     

Synthesis, structural characterization and antibacterial activity of 2,6-diacetylpyridine bis(benzenesulfonohydrazide) Schiff bases and their copper(II) complexes

2,6-Diacetylpyridine bis(benzenesulfonohydrazide) Schiff bases (L¹, L² and L³) and their Cu(II) complexes of the general formula [CuL·H₂O] were synthesized and characterized by various spectroscopic techniques. The crystal structure of [CuL³·(py)]·py was investigated by single crystal X-ray structure analysis. The Cu(II) cation has near square pyramidal, penta-coordinate geometry. The binegatively charged tetradentate Schiff base is asymmetrically coordinated to the Cu(II) ion via the pyridine N atom, the azomethine N atom, the sulfonyl O atom and the deprotonated hydrazine N atom. There is a pyridine molecule apically coordinated to the Cu(II) ion. All the Schiff bases and their copper(II) complexes were screened by the disc diffusion method against multi-drug resistant (MDR) gram-negative and gram-positive bacteria. The minimum inhibitory concentration (MIC) values were also determined. These results show that the antibacterial activity of the Schiff bases against Methicillin-resistant Staphylococcus aureus (MRSA) is enhanced when they are chelated with the copper(II) ion.     

耐有机溶剂微生物及酶类在生物催化中的应用*

将生物催化剂运用到有机相催化具有多种优势,然而酶是一类不稳定的催化剂很容易在有机溶剂存在的条件下失去活性。耐有机溶剂微生物是一类新颖的极端微生物,它们可以在高浓度的有机溶剂存在的条件下生存,而这类微生物所产的一些酶类往往具有耐有机溶剂的性能。随着这一领域开发和发展,从这类极端微生物中寻找高效生物催化剂逐渐成为新的研究热点,并被越来越多的运用到有机相生物催化领域。本文综述了新型耐有机溶剂催化剂的开发、应用与发展,并讨论了该领域的研究所带来的机遇与挑战。     

甲烷氧化细菌催化二氧化碳生物合成甲醇的研究

甲烷氧化细菌中包含的甲烷单加氧酶(MMO)、甲醇脱氢酶(ADH)、甲醛脱氢酶(FaldDH)、甲酸脱氢酶(FateDH)经过一系列反应能够把甲烷深度氧化生成二氧化碳，并生成一定的能量物质．把二氧化碳还原为甲醇是一个需要能量的过程，目前还没有已知的有机体在温和条件下完成这一反应．研究发现，甲基弯菌Methylosi-nus trichosporium IMV 3011可以催化二氧化碳生物转化生成甲醇．在休眠的悬浮细胞中充人二氧化碳后，反应一段时间在反应液中检测到了甲醇．二氧化碳转化成甲醇是一个需要能量推动的反应，为了补充反应所消耗的能量．反应一段时间后需要用甲烷进行再生，以恢复细胞中的还原当量NADH．我们进行了反应再生的交替连续批式反应，甲醇积累量能够维持在一个比较稳定的水平．理论上，反应不会增加温室效应，这是一个有效的、环境友好的、可恢复的反应过程．     

Supercapacitors Based on c‐Type Cytochromes Using Conductive Nanostructured Networks of Living Bacteria

Supercapacitors have attracted interest in energy storage because they have the potential to complement or replace batteries. Here, we report that c‐type cytochromes, naturally immersed in a living, electrically conductive microbial biofilm, greatly enhance the device capacitance by over two orders of magnitude. We employ genetic engineering, protein unfolding and Nernstian modeling for in vivo demonstration of charge storage capacity of c‐type cytochromes and perform electrochemical impedance spectroscopy, cyclic voltammetry and charge–discharge cycling to confirm the pseudocapacitive, redox nature of biofilm capacitance. The biofilms also show low self‐discharge and good charge/discharge reversibility. The superior electrochemical performance of the biofilm is related to its high abundance of cytochromes, providing large electron storage capacity, its nanostructured network with metallic‐like conductivity, and its porous architecture with hydrous nature, offering prospects for future low cost and environmentally sustainable energy storage devices.     

Characterization of the Core Oligosaccharide and the O‐Antigen Biological Repeating Unit from Halomonas stevensii Lipopolysaccharide: The First Case of O‐Antigen Linked to the Inner Core

A novel core structure among bacterial lipopolysaccharides (LPS) that belong to the genus Halomonas has been characterized. H. stevensii is a moderately halophilic microorganism, as are the majority of the Halomonadaceae. It brought to light the pathogenic potential of this genus. On account of their role in immune system elicitation, elucidation of LPS structure is the mandatory starting point for a deeper understanding of the interaction mechanisms between host and pathogen. In this paper we report the structure of the complete saccharidic portion of the LPS from H. stevensii. In contrast to the finding that the O‐antigen is usually covalently linked to the outer core oligosaccharide, we could demonstrate that the O‐polysaccharide of H. stevensii is linked to the inner core of an LPS. By means of high‐performance anion‐exchange chromatography with pulsed amperometric detection we were able to isolate the core decasaccharide as well as a tridecasaccharide constituted by the core region plus one O‐repeating unit after alkaline degradation of the LPS. The structure was elucidated by one‐ and two‐dimensional NMR spectroscopy, ESI Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometry, and chemical analysis.